Richard Ramsden

Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens

Many diverse human diseases are associated with protein aggregation in ordered fibrillar structures called amyloid. Amyloid formation may mediate aberrant protein interactions that culminate in neurodegeneration in Alzheimer, Huntington, and Parkinson diseases and in prion encephalopathies. Studies of protein aggregation in the brain are hampered by limitations in imaging techniques and often require invasive methods that can only be performed postmortem. Here we describe transgenic mice in which aggregation-prone proteins that cause Huntington and Parkinson disease are expressed in the ocular lens. Expression of a mutant huntingtin fragment or α-synuclein in the lens leads to protein aggregation and cataract formation, which can be monitored in real time by noninvasive, highly sensitive optical techniques. Expression of a mutant huntingtin fragment in mice lacking the major lens chaperone, αB-crystallin, markedly accelerated the onset and severity of aggregation, demonstrating that the endogenous chaperone activity of αB-crystallin suppresses aggregation in vivo. These novel mouse models will facilitate the characterization of protein aggregation in vivo and are being used in efficient and economical screens for chemical and genetic modifiers of disease-relevant protein aggregation.

Determining Protein Complex Structures Based on a Bayesian Model of in Vivo Förster Resonance Energy Transfer (FRET) Data

The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking.

A large insertion in the Shiga-like toxin 2 gene (stx2) of an Escherichia coli O157:H7 clinical isolate

Abstract Six clinical Escherichia coli O157:H7 isolates were epidemiologically linked as part of an outbreak in which the most likely source was undercooked ground beef or cross-contamination from the ground beef to other food products at a Mexican-style restaurant. These cultures were analyzed using molecular genetic, immunological and cytotoxicity procedures. All six isolates were confirmed as E. coli O157:H7 and were indistinguishable by pulsed-field gel electrophoresis using XbaI. The results of polymerase chain reaction (PCR) tests, non-isotopic gene probing, reversed passive latex agglutination (RPLA) kit results and Vero cell assays were consistent for the presence of a functional Shiga-like toxin 1 (Stx 1) protein. All six strains produced a stx2 PCR amplicon product; five strains produced a product which was consistent with the predicted amplicon size and one (SEA 6414) produced a much larger PCR product. The SEA 6414 isolate produced a protein reactive with the RPLA kit anti-Stx 2 antibody but was not cytotoxic to Vero cells. Sequencing of this region revealed that this 1310 bp insertion was very similar to a previously identified IS 1203 sequence and the insertion interrupted the carboxyl end of the coding region of the stx2 gene ‘A’ subunit.

An intein with genetically selectable markers provides a new approach to internally label proteins with GFP

BackgroundInteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein.ResultsThe Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1) aminoglycoside phosphotransferase; 2) imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3) hygromycin B phosphotransferase; and 4) the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene CMD1. The DNA was transformed into yeast and integrants obtained by direct selection for the inteins marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker.ConclusionsInteins continue to show their flexibility as tools in molecular biology. The Pch PRP8 intein can successfully tolerate a variety of genetic markers and still retain high splicing efficiency. We have shown that a genetically marked intein can be used to insert GFP in one-step within a target protein in vivo.

Dual NRF2 paralogs in Coho salmon and their antioxidant response element targets

The transcription factor NFE2L2 (Nuclear Factor, Erythroid 2-Like 2, or NRF2) plays a key role in maintaining the redox state within cells. Characterization of this pathway has extended to fish, most notably zebrafish (Danio rerio), in which two paralogs of the transcription factor exist: Nrf2a, an activator, and Nrf2b, a negative regulator during embryogenesis. Only one ARE target has been thoroughly delineated in zebrafish, and this deviated from the canonical sequence derived from studies in mammals. In general, the mechanistic pathway has not been characterized in non-model aquatic organisms that are commonly exposed to environmental pollutants. The current study compares the zebrafish paralogs to those found in a non-model teleost, the ecologically important salmonid, Oncorhnychus kisutch (coho salmon). Two salmon paralogs, Nrf2A and -2B, described here were found to possess only slightly greater identity between one another (84% of amino acids) than to the singleton ortholog of the esocid Esox lucius (80–82%), the nearest non-salmonid outgroup. Unlike one of the zebrafish forms, each is a strong activating factor based on sequence homology and in vitro testing. To uncover functional target AREs in coho, promoter flanking sequences were isolated for five genes that protect cells against oxidative stress: heme oxygenase 1, peroxiredoxin 1, glutamate-cysteine ligase, and the glutathione S-transferases pi and rho (hmox1, prdx1, gclc, gstp, and gstr). All except gstr had functional elements and all fit the standard mammalian-derived canonical sequence, unlike the motif found in zebrafish gstp. Expression studies demonstrate the presence of both Nrf2 paralogs in multiple organs, although in differing ratios. Collectively, our findings extend the conservation of Nrf2 and the ARE to salmonids, and should help inform future work in teleosts on mechanisms of redox control, as well as responsiveness of this pathway and its downstream antioxidant gene targets to chemical exposures in the environment.

Isolating cytochrome P450 cDNA and genomic clones: library screening with synthetic DNA oligomers.

Publisher Summary This chapter discusses the isolating cytochrome P450 complementary DNA (cDNA) and genomic clones. Cytochrome P450 enzymes are encoded by a large and complex superfamily of genes. Current P450 gene pools are believed to be the products of extensive duplication events, ultimately descended from one or more ancestral genes. Notable regions of conservation and divergence are apparent within P450 structures. Amino acid sequence conservation is found near a carboxy-terminal cysteine residue; this region is thought to function as the ligand to heme at the enzyme active site. Traditional methods of library screening fall within two categories: (1) antigenic detection using antibodies, and (2) homology to radiolabeled nucleic acid probes. Identification of target cDNAs by the interaction of antigens with antibody probe requires that the cloned DNA be inserted into an expression vector; the insert DNA must be in the correct orientation and reading frame.